chapter 3 immobilized enzymes

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  • ImmobilizationImmobilized biocatalysts can be made from killed cells, cell fragments, purified enzymes, and respiring cellsDefinition: Enzymes or cells which are physically confined to a defined region in space while retaining their catalytic activity and have the ability to be repeatedly and continuously used

  • Immobilized Enzyme SystemsEnzyme Immobilization:

    - Easy separation from reaction mixture, providing the ability to control reaction times and minimize the enzymes lost in the product.

    - Re-use of enzymes for many reaction cycles, lowering the total production cost of enzyme mediated reactions.

    - Ability of enzymes to provide pure products.

    - Possible provision of a better environment for enzyme activity

    - Diffusional limitation

  • Higher dilution rates can be used in a CSTR. Easier product recovery. Lowered viscosity.Easier enzyme recovery.

    Why.Advantages of Immobilization

  • Mass transfer is inhibitedReduced activity of enzymesBursting of immobilization medium due to cell growthCells/enzymes can leak out of immobilization medium Disadvantages of Immobilization

  • Immobilization MethodsPhysicalChemical

  • Membranes

  • Membrane MicroencapsulationMembrane polymerized around aqueous enzyme solution in colloidal suspension (particle sizes on the order of 100-10 m)

    Organic solventAqueous enzyme solutionmixingAdd polymer

  • MicroencapsulationAdvantagesHigh surface to volume ratioThin membraneRelatively benign attachment methodProblemsMore easily damaged than gel beads

  • Other Membrane Immobilization Methods- HFMHollow fiber reactors use micro- or ultra-filtration membranes to retain high MW enzymes, but pass low MW compoundsEnzymes or cellsSubstrate and products outside the membraneSubstrate diffuses through membraneProduct diffuses out of the membrane

  • Close-up view of fiber

  • Hollow Fiber ReactorEnzymes or cells inSubstrate inProduct out

  • Two Types of Cartridges

  • Entrapment

  • Entrapment Immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane. - retain enzyme - allow the penetration of substrate.

    It can be classified into matrix and micro capsule types. Immobilized Enzyme Systems

  • Immobilized Enzyme SystemsEntrapment - Matrix Entrapment- Membrane Entrapment(microencapsulation)

  • EntrapmentAdvantagesRelatively benign attachment methodEasy to performProblemsMass transfer limitationsCell/enzyme leakageSome cell/enzyme deactivationAlginate (polymer) common entrapment matrix

  • Immobilized Enzyme SystemsMatrix Materials: Organics: polysaccharides, proteins, carbon, vinyl and allyl polymers, and polyamides. e.g. Ca-alginate, agar, K-carrageenin, collagenImmobilization procedures:Enzyme + polymer solution polymerization extrusion/shape the particles

    Inorganics: activated carbon, porous ceramic.

    Shapes: particle, membrane, fiber

  • Immobilized Enzyme SystemsEntrapmentchallenges:- enzyme leakage into solution

    - diffusional limitation

    - reduced enzyme activity and stability

    - lack of control micro-environmental conditions.

    It could be improved by modifying matrix or membrane.

  • Covalent Bonding

  • Immobilized Enzyme SystemsSurface immobilizationAccording to the binding mode of the enzyme, this method can be further sub-classified into: - Physical Adsorption: Van der WaalsCarriers: silica, carbon nanotube, cellulose, etc. Easily desorbed, simple and cheap, enzyme activity unaffected.

    - Ionic Binding: ionic bonds Similar to physical adsorption.Carriers: polysaccharides and synthetic polymers having ion-exchange centers.

  • Immobilized Enzyme SystemsSurface immobilizationCovalent Binding: covalent bonds Carriers: polymers contain amino, carboxyl, sulfhydryl, hydroxyl, or phenolic groups.

    - Loss of enzyme activity - Strong binding of enzymes

  • Covalent BondingMost extensively used method of immobilization due to high bond strengthThree elements- structural polymer, enzyme molecule and bridge moleculeActivity of an immobilized enzyme is a strong function of the hydrophilicity of the structural polymer

  • Covalent Bonding To SupportBridging molecule needs to be small, have two reactive groups, and not bond at the active site

  • Immobilized Enzyme SystemsCross-linking is to cross link enzyme molecules with each other using agents such as glutaraldehyde.

    Features: similar to covalent binding.

    Several methods are combined.

  • Copolymerization of EnzymesCopolymerization is performed with multifunctional bridge molecules to yield and insoluble productUsually an inert protein is also included

  • Chemistry of Covalent Immobilization

  • Adsorption

  • Immobilization by AdsorptionBinding forces are ionic, hydrophobic, hydrogen bonds, or Van der Waals interactionsBinding is simple (stir together in a beaker) but is reversible. Substrate addition can cause desorption.

  • Typical AdsorbentsCellulosePolystyrene resinsKaoliniteGlassAluminaSilica gel